Protein markers play a crucial role in proteomics research with the coming of post-genome era. However, protein markers nowadays still have many restrictions and inconveniences. For example, (1) currently, the protein markers have to be copied manually from nitrocellulose paper to film instead of developing directly in the film during western blotting; (2) the common pre-stain protein markers sold on market have low-accuracy since the staining results in heterogeneity and electricity alteration; and (3) the current protein marker kits use known proteins as markers, yet their molecular weights are fixed and irregular. Therefore, there is a need for developing an auto-developing and regularly weighted protein molecular weight marker for Western blot to solve the problems encountered in proteomics research.
There are presently various types of protein markers for electrophoresis and Western blotting, and most of them are pre-stain markers. For instance, the multicolored protein marker is known for its colorful marker that enables easy observation, but the low-accuracy problem is still unsolved.
Chang et al. used a set of green fluorescent protein (GFP) fused proteins to construct dye-free protein molecular weight markers, which can emit fluorescence and present bands as regular as a ladder (Chang M., Hsu H. Y. and Lee H. J. Dye-free protein molecular weight markers. Electrophoresis, 26: 3062-68, 2005). Although the markers are convenience, they cannot be heated since GFP would be denatured and lose function. Without heating, however, the markers cannot be denatured thoroughly. Thus, the low-accuracy problem still remains.
Biotinylated protein markers are also available. These markers are dye-free but additional biotin label is required. Moreover, in order to be detected by color reaction, there is a need of labeling with HRP-conjugated anti-biotin antibody or HRP-conjugated avidin, which causes many inconveniences. In addition, biotin labeling may also alter the electric charge of protein marker and result in inaccuracy.
There are products of HIS-tag, S-tag or E-tag-fused protein markers as well. When using HIS-tag, S-tag, or E-tag antibodies to carry out development, the protein markers would auto-develop on film simultaneously. Those protein markers are not popular for the reason that the color presents simultaneously only when HIS-tag, S-tag, or E-tag antibodies is used to monitor protein expression. Otherwise, adding HRP-conjugated HIS-tag, S-tag, or E-tag antibodies is needed to activate the color reaction, which makes the procedure quite troublesome.